Effects Of SO₂ Exposure On Mouse Lung And Alleviation Effect Of The Exopolysaccharide From Bacillus Subtilis

Sulfur dioxide（SO₂）is a common air pollutant,which is mainly caused by the burning of fossil energy.With the process of industrialization and urbanization,a large number of SO₂emissions have caused serious air pollution.At present,the concentration of SO₂ is still very high in some parts of developing countries,factories,mines and other specific areas,which has a great impact on people’s health.SO₂ can destroy the structure and stability of respiratory system,and cause respiratory tract inflammation and other diseases.Its research mainly focused on pathology,biochemical indicators,genotoxicity,oxidative stress and DNA damages,but the molecular mechanism of its lung damage is rarely reported.In addition,SO₂ can aggravate respiratory diseases of asthma.Asthma,as a chronic airway inflammatory disease,is caused by allergens and air pollutants,and involves inflammation and immune disorders.SO₂ can induce and enhance asthma symptoms and susceptibility,and its related researchs are mainly concentrated in the field of epidemiology,but its molecular mechanism research is less,lacking direct experimental evidence.The toxicity of SO₂ to the body is generally believed to be related to the increase of reactive oxygen species and the induction of inflammation.At present,there is no effective means of prevention and treatment.Exopolysaccharide（EPS）isolated from Bacillus subtilis,as an active substance of probiotics,has the effects of antioxidation and enhancing immunity.However,there is no report about whether EPS can alleviate respiratory diseases induced by SO₂.Aiming at the above problems,this paper puts forward the research of this topic.In this paper,the effects of different concentrations of SO₂ and different exposure modes on lung damage and inflammation were studied,and the toxicity of SO₂ derivatives on airway epithelial cells and alveolar macrophages was detected in vitro.Then,the aggravating effect and mechanism of SO₂ on allergic asthma were further discussed.In addition,according to the toxicity of SO₂,the structure and biological activity of extracellular polysaccharide from Bacillus subtilis were analyzed,as well as its effect and mechanism of alleviating SO₂-induced lung damage in mice.The main experimental results are as follows:Firstly,in this study,the effects of acute SO₂ exposure on body weight,lung-to-body ratio,pathological changes of lung,the number of inflammatory cells in alveolar lavage fluid（BALF）,oxidative damage of lung,inflammatory cytokines,anti-inflammatory cytokines,mucin5AC,remodeling factors,apoptosis genes,expression of protocarcinoma and tumor suppressor genes and their mechanisms were studied.The results showed that SO₂ exposure could reduce the weight of mice,resulting in the increase of lung-body ratio and immune organs（spleen of thymus）.It can also induce inflammatory cell infiltration in lung tissue,bronchial epithelial thickening,mucus secretion and airway stenosis.In addition,the number of lymphocytes,eosinophils and macrophages in alveolar lavage fluid（BALF）exposed to SO₂ increased significantly.The contents of H₂O₂,GSH and MDA in lung were significantly increased,and the activity of SOD was significantly decreased.Although the changes of oxidative stress in serum were changed,there was no statistical significance.The expression of TNF-α,IL-1βand IL-6 in lung was significantly increased,the expression of IFN-γwas significantly decreased,and the expression of four anti-inflammatory cytokines（IL-4,IL-5,IL-13 and TGF-β）was significantly up-regulated.SO₂ exposure significantly increased the expression of mucin MUC5AC gene;the m RNA expression of Fibronectin,matrix metalloproteinase-9（MMP-9）,smooth muscle calmodulin and smooth muscle myosin（sm MHC）were significantly increased.SO₂exposure significantly increased the pro-apoptotic gene Bax,caspase-3 and Bax/Bcl-2,and significantly down-regulated the transcription of anti-apoptotic gene Bcl-2;the expression of proto-oncogenes（C-fos and C-jun）and tumor suppressor gene p53 was significantly increased,and the m RNA expression of tumor suppressor gene Rb was significantly decreased.To sum up,acute SO₂ exposure can cause lung airway damage,mucus secretion,inflammation and secretion of anti-inflammatory factors,induce airway remodeling,and promote apoptosis and canceration.Secondly,in order to clarify the damage effect and mechanism of SO₂ on lung cells,the effects of SO₂ derivatives on the survival rate,ROS level,cytokines,transcription factors,membrane receptors,apoptosis and the expression of protocarcinoma and tumor suppressor genes of airway epithelial cells and alveolar macrophages in vitro were further evaluated.The results showed that with the increase of SO₂ derivative concentration,the survival rate of epithelial cells and macrophages decreased,and there was a significant difference at the concentration of 1 m M;0.1 m M SO₂ derivatives significantly increased the ROS level of cultured cells in vitro,and increased the m RNA expression of mucin MUC5AC,transforming growth factorβ（TGF-β）,epidermal growth factor（EGF）and interleukin-6（IL-6）in airway epithelial cells.In addition,TGFR and TLR2 receptor,NF-κB and STAT6 transcription factor m RNA in airway epithelial cells were significantly increased.The expression of Bax and Caspase-3 genes,proto-oncogene C-fos and tumor suppressor gene Rb were significantly increased.In addition,SO₂ derivatives significantly increased the pro-inflammatory factors（IL-6,IL-1β,TNF-αand INF-γ）secreted by pulmonary macrophages,and the expressions of inflammatory pathways related to TLR4/My D88,JAK1 and NF-κB were activated,while the expression of Bcl-2 m RNA decreased.These results suggest that the toxicity of SO₂ to airway epithelial cells may activate TGFR and TLR2 signaling pathways through oxidative stress,release cytokines,promote the proliferation and fibrosis of epithelial cells,and induce inflammation,apoptosis and carcinogenesis.SO₂ may induce the immune response and inflammatory reaction by activating TLR4/My D88 and JAK1 pathways in alveolar macrophages.Thirdly,the asthmatic mouse model induced by ovalbumin（OVA）was established,and the effects of SO₂ on inflammation in asthmatic mice were detected by lung pathological analysis,white cell count in alveolar lavage fluid（BALF）and quantitative PCR.The results showed that SO₂ increased the number of lymphocytes,macrophages and eosinophils in BALF in asthmatic mice.The expression levels of Th2 cytokines（IL-4,IL-5,IL-13）,IL-6 and MUC5AC were significantly increased.The levels of malondialdehyde（MDA）and glutathione（GSH）were increased,the activity of superoxide dismutase（SOD）was decreased,and the level of reactive oxygen species（ROS）was obviously increased.The expressions of TLR4/NF-κB and TCR/GATA-3pathways were significantly up-regulated and the levels of NF-κB and^[P]ERK1/2transcription factors were enhanced.The above results indicated that SO₂ may increase the number of macrophages and lymphocytes,increase the level of ROS,activate TLR4 and TCR pathways,and thus increased the expression of Th2 cytokines to enhance the eosinophil inflammation in asthma.Finally,studies have confirmed that the main mechanism of lung damage caused by SO₂ is oxidative stress and inflammation.It is suggested that the lung damages caused by SO₂ may be alleviated by antioxidation and enhancing immunity.In this experiment,the groups contained in extracellular polysaccharide（EPS）produced by Bacillus subtilis and its antioxidant and tumor cell inhibitory activities in vitro were identified,and then the effect of EPS on lung damage induced by SO₂ was evaluated in mice.The results showed that the structure of exopolysaccharide contained O-H group,C=C,C=O,CH₃,C-O-C bond andα-pyranose,which had excellent oxygen free radical scavenging rate and cancer cell inhibition rate in vitro.EPS can significantly improve the immune organ index of mice,and reduce the weight loss and pulmonary edema caused by SO₂.The number of total inflammatory cells,lymphocytes,eosinophils and macrophages in BALF decreased significantly.The levels of H₂O₂ and MDA were significantly reduced,and the activities of SOD and GSH-PX were significantly increased.The expression of MUC5AC,pro-inflammatory factors IL-6 and TNF-α,inflammatory pathway STAT6 and NF-κB genes in lung tissue were significantly down-regulated.The expression of apoptosis indexes Bax/Bcl-2 and Caspase-3 were significantly decreased,and the expression of Cyclin B1 was significantly increased.The expression of proto-oncogenes（C-fos and C-jun）was significantly down-regulated,and the expression of tumor suppressor genes（p53 and Rb）was significantly up-regulated,so that all indexes basically recovered to the level of the control group.These results suggest that EPS may alleviate SO₂-induced lung injury by alleviating SO₂-induced lung oxidative injury,inflammatory reaction,apoptosis and canceration,and improving immune function,and play protective roles in SO₂-induced lung injury in mice.In summary,acute SO₂ exposure can cause structural damage of lung tissue in mice,infiltration of inflammatory cells,and activation of inflammatory pathways and secretion of inflammatory factors,induce airway remodeling,promote apoptosis and canceration.SO₂ exposure may also aggravate asthma symptoms by strengthening lung tissue damage such as inflammation,airway remodeling and apoptosis.SO₂ derivatives can induce airway epithelial cells to secrete mucus and inflammatory factors,and cause airway remodeling and canceration.SO₂ derivatives can also induce inflammatory reaction and apoptosis of alveolar macrophages.After EPS intervention,the lung damage caused by SO₂ exposure can be alleviated by reducing the oxidative damage,inflammatory response,regulating apoptosis,protocarcinoma and tumor suppressor genes.This study can provide its health risks and experimental basis for the prevention and treatment of lung damage caused by SO₂.